A tubular structure, which is reversibly formed by self-assembly in water of accurately designed amphiphilic molecules, encapsulates a hydrophilic guest such as biopolymer, protein (natural state), DNA, various nanoparticles, as well as small molecules, into a hydrophilic hollow cylinder having an inner diameter of 5 to 100 nm. The tubular structure is easily dispersed in water due to outer and inner surfaces covered with a hydrophilic group. In addition, the tubular structure as described above controls storage and release of the encapsulated hydrophilic guest in response to external stimulation such as pH, temperature, light, additive, and thereby the tubular structure has been particularly paid attention in a biotechnological or medical field (Non-Patent Literature 1). It is expected to apply the tubular structure to an immobilization matrix or the like a storage capsule of proteins, or a drug delivery capsules, using characteristics of the tubular structure (Non-Patent Literatures 2 and 3). However, in most of the existing organic nanotubes, since inner and outer surfaces thereof were covered with the same hydrophilic portions, it was difficult to selectively and efficiently encapsulate a raw material such as a drug, protein in the organic nanotube (Non-Patent Literatures 4 to 6). In detail, during encapsulation of the raw materials, a complicated operation has been required such that removing water in the organic nanotube by freeze-drying under reduced pressure, or the like, and adding an aqueous solution in which the raw materials are dispersed and dissolved to thereby rehydrate the organic nanotube (so called capillary phenomenon) (Non-Patent Literatures 4 and 5).
The present inventors have, so far, developed a manufacturing technology of an organic nanotube (asymmetric organic nanotube), having a monolayer membrane structure in which inner and outer surfaces thereof are covered with respective hydrophilic groups, by self-assembly in water of asymmetric bipolar lipid molecules having two different hydrophilic groups at both ends of an hydrophobic alkylene chain (Patent Literature 1, and Non-Patent Literature 1). Recently, the present inventors developed and filed a patent for an asymmetric nanotube capable of slowly releasing a drug and having characteristics as an excellent drug capsule by encalulating an amino amino-group containing drug such as doxorubicin, using an asymmetric bipolar lipid molecules in which 2-glucosamine and oligo glycine moieties are linked to both ends of long chain dicarboxylic acid via amide bonds, respectively (Japanese Patent Application No. 2010-194544). Since a functional group capable of interacting with a hydrophilic guest may be site-specifically localized on the inner surface of the asymmetric organic nanotube, the hydrophilic guest may be selectively and efficiently encapsulated in a hollow cylinder of the asymmetric organic nanotube. In addition, release of the hydrophilic guest into the bulk may be controlled by applying external stimulus to reduce interactions between the inner surface of the asymmetric organic nanotube and the hydrophilic guest. For example, a small molecule, oligo DNA, double stranded DNA, protein, nanoparticles, or the like may be selectively and efficiently encapsulated as an anionic and hydrophilic guest in a hollow cylinder having a cationic property by protonation of an amino group localized on the inner surface of the asymmetric nanotube. The encapsulated hydrophilic guest may be sustainably released into the bulk by applying external stimulus to change a pH in a solution so as to perform deprotonation of the amino group on the inner surface of the asymmetric organic nanotube.
It has been demanded to develop an encapsulant capable of encapsulating the drug simply and efficiently, maintaining stably, and continuously releasing a drug in target cells of lesions such as cancer cells or tissue. Particularly, since anthracycline based anticancer agents, such as doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin, widely used for treating cancer have strong toxicity, particularly, cardiac toxicity, it has been demanded to develop an excellent encapsulant capable of directly and effectively delivering the anticancer agent to cancer cells and sustainably-releasing the anticancer agent. In addition, in view of reducing an administration frequency or dose of a drug to thereby reduce burden of patients, it has been urgently demanded to develop a technology of controlling a sustained-release rate of a drug.
In the case of a liposome based drug nanocapsules (Non-Patent Literature 7) prepared in this regard, since a pH gradient was used, subsequent purification was required. Therefore, preparation was complicated and required a long period of time.
In contrast, the asymmetric nanotube, developed by the present inventors (Japanese Patent Application No. 2010-194544, or the like), has a carboxylic-acid inner surface, and is thereby-capable of encapsulating a hydrophilic guest including the amino group-containing drug doxorubicin, or the like in a hollow cylinder by mixing the nanotube and the drug in water because the nanotube has a carboxylic-acid inner surface. In addition, the asymmetric nanotube has a sustained-release function depending on a change in environmental pH. Therefore, the asymmetric nanotube is effectively applied as a drug capsule.
However, in the case of the asymmetric nanotube according to the related art developed by the present inventors, there was no method of precisely controlling a sustained-release property of the encapsulated drug under physiological conditions. In addition, since there was a problem in stability of the tube itself as describe below, a precisely controlling the sustained-release was not yet achieved.
Particularly, in a carboxylic acid based asymmetric bipolar lipid group according to the related art, the resultant asymmetric nanotube was slowly changed into another morphology, such that a sustained-release property of the drug was lost after being exposed for only 2 hours or for at most about 12 hours under physiological conditions. More particularly, an asymmetric nanotube (Non-Patent Literature 8) formed by lipid molecules in which a 1-glucopyranosyl group was bonded at one end of long chain dicarboxylic acid via an amide bond was slowly changed, under the physiological conditions (for example, PBS buffer, pH 7.5), into a microtube having an outer diameter of 120 to 200 nm, an acicular crystal, or the like, after about 4 hours. Further, during a manufacturing process of the asymmetric nanotube according to the related art, since microtubes or tape-like assemblies were simultaneously formed, a separation and purification process of the nanotube by centrifugation, or the like, has been required. In addition, there were problems in that at the time of long term preservation at room temperature or a cold temperature, the asymmetric nanotube may be easily crystallized, and long term preservation stability was not high.
A development of asymmetric bipolar lipid molecules in which mono- and di-glycine residues are introduced at a carboxylic acid terminus of the carboxylic acid based lipid molecule enabled us to solve these problems described above, such as lack of long term preservation stability of the nanotubes, and requirement of separation and purification process (Non-Patent Literatures 9 and 10). Further, asymmetric nanotubes formed by these lipid molecules were able to improve a long-term preservation property. However, in these asymmetric nanotubes, there was a problem such that after being exposed under physiological conditions (PBS buffer, pH 7.5), an inner diameter of the nanotube was changed from about 7 nm into about 50 nm (it will be described below in Example 5).
Even in an asymmetric organic nanotube (Japanese Patent Application No. 2010-194544) recently developed by the present inventors, the problem such that after being exposed under physiological conditions for several hours, a structure of the asymmetrical nanotube was changed into a fibrous structure was not solved. Hence, a sustained-release property at the time of administering the nanotube to a body as a drug capsule should be further improved, and it is necessary to precisely control the sustained-release.
In addition, as described above, since a target drug in a delivery system for directly delivering the drug to the lesion site, such as the anticancer drugs including doxorubicin, or the like, often has high hydrophobicity, it is necessary to use an encapsulant capable of delivering a hydrophobic guest to the lesion site and slowly releasing the hydrophobic guest in the lesion site.
As a technology of encapsulating a hydrophobic molecule according to the related art, only in the case of a hydrophobic small molecule capable of being encapsulated in a cyclodextrin hollow pore having a size of about 1 nm, the hydrophobic small molecule may be encapsulated in a hydrophilic hollow cylinder of an organic nanotube formed by amphiphilic molecules and released in water by forming a complex with cyclodextrin (Patent Literature 2). However, in order to prepare the complex, a complicated process was required, and a yield thereof was low. In addition, since the organic nanotube disclosed in the corresponding document has a bilayer structure, inner and outer surfaces thereof are covered with the same hydrophilic group. Thus, the cyclodextrin complex may not be selectively encapsulated in the hollow cylinder. Further, since there is no specific interaction between the inner surface of the hollow cylinder and the cyclodextrin complex, it is impossible to control release.
An intercalated-type organic nanotube which are able to decentrally embed hydrophobic small molecules having a size of 1 to 3 nm in a membrane wall of the organic nanotube was developed (Patent Literature 3). The hydrophobic small molecules may be embedded at a content of 10% based on substrate lipid molecules and dispersed in water while maintaining a shape of the organic nanotube. However, in order to allow the hydrophobic small molecules embedded in the membrane wall to be released, the organic nanotube should be decomposed using an additive, or the membrane wall should be changed in a fluid state by heating the organic nanotube at a gel-liquid crystal phase transition temperature or more (Non-Patent Literature 11). In this case, the hydrophobic small molecules may be relatively rapidly released.
Although an organic nanotube of which all of the inner and outer surfaces are covered with a hydrophobic group was developed, hydrophobic small molecules, hydrophobic nanoparticles, or the like, are adsorbed in the inner and outer surfaces, but they are mainly adsorbed in the outer surface (Patent Literature 4), thus this organic nanotube may not disperse hydrophobic molecules in water.
As described above, various fields have been, therefore, urgently demanded to develop an organic nanotube which has a hydrophilized outer surface and a hydrophobized outer surface, capable of having a sufficient space in a hydrophobic hollow cylinder, enabling direct interaction between the inner surface and a hydrophobic guest, and capable of releasing the hydrophobic guest while maintaining a shape of the organic nanotube which has not yet been implemented. If the organic nanotube as described above is constructed, it will be possible to selectively encapsulate a hydrophobic polymer and a hydrophobic nanostructure as well as a hydrophobic small molecule in the hollow cylinder to disperse the encapsulated material in water. In addition, unlike the related art (Patent Literature 2), since the inner surface of the nanotube and the hydrophobic guest may directly interact with each other, if the interaction is suppressed through external stimulus, release control is possible. Further, the hydrophobic guest may be released even without decomposing the organic nanotube or making the membrane wall to be in a fluid state as in the related art (Patent Literature 3).
In addition, the above-described asymmetric organic nanotube (Japanese Patent Application No. 2010-194544) recently developed by the present inventors, in the case of using hydrophobic guests, for example, a drug having high hydrophobicity such as doxorubicin, a sufficient sustained-release property was not obtained, and in view of precisely controlling the sustained-release, the asymmetric organic nanotube was not sufficient.
However, it also has been demanded to develop, with regard to encapsulation of hydrophobic molecules, an efficient method of refolding genetic recombinant protein, or the like.
That is, it has been urgently demanded, in view of industrial mass-production of normal protein, to develop a material having a nano space for encapsulating of protein in which hydrophobic groups are partially exposed by denaturation (denatured protein). β-zeolite, which is a porous inorganic material, may adsorb denatured protein (Non-Patent Literature 12). It was reported that protein was separated from β-zeolite by cleaning a denaturant and adding a buffer solution containing polyethylene glycol or surfactant to thereby be recovered (refolded) to have a normal structure. In addition, an amphiphilic polymer in which a cholesteryl group as a hydrophobic group is introduced in polysaccharide, which is a hydrophilic polymer, may be self-assembled in water to form a gel (nanogel) having a size of 20 to 30 nm (Non-Patent Literature 13). It was reported that the denatured protein was encapsulated in the inner space of the nanogel, then release and refolding of protein were simultaneously occurred when the nanogel was collapsed by adding cyclodextrin (Non-Patent Literature 14). However, a protein solution obtained from the β-zeolite and the nanogel includes polyethylene glycol, or the surfactant, cyclodextrin, and a composite polymer of cyclodextrin and hydrophobized polysaccharide, and a complicated separation process for removing other components except for the protein is required.
Therefore, it has been strongly expected to develop an organic nanotube of which only an inner surface of a hollow cylinder portion thereof is hydrophobized (an organic nanotube having a hydrophobized inner surface) in order to solve all problems as described above. That is, it has been urgently demanded to develop the organic nanotube having a hydrophobized inner surface capable of selectively and efficiently encapsulating denatured protein as a hydrophobic guest, having a function of assisting in refolding, and releasing normal protein without adding a specific additive. In addition, it has been urgently demanded to develop the organic nanotube having a hydrophobized inner surface capable of selectively and efficiently encapsulating a drug such as doxorubicin as a hydrophobic guest and sustainably-releasing the drug into a bulk according to the external environment.
Particularly, in controlling sustainable-release of the drug, it has been strongly expected to provide an asymmetric nanotube capable of maintaining a tubular structure for a long period of time even under any physiological conditions (for example, in PBS buffer at pH 7.5 and 35° C.) while maintaining the existing excellent properties of stable carboxylic acid based asymmetric nanotube groups having an inner surface covered with a carboxylic group, and a new asymmetric bipolar lipid molecule for the asymmetric nanotube. That is, it has been urgently demanded to provide an asymmetric bipolar lipid molecule capable of manufacturing a nanotube capsules with excellent properties as follows: selectively and efficiently preparing an asymmetric nanotube with a high yield under mild conditions; having long-term preservation stability in the obtained asymmetric nanotube; efficiently encapsulating a cationic drug such as doxorubicin at a high concentration; and having stable morphology of the tube under physiological conditions.